Anne-Sophie Walker

Fungicide-driven evolution and molecular basis of multidrug resistance in field populations of the grey mould fungus Botrytis cinerea

The grey mould fungus Botrytis cinerea causes losses of commercially important fruits, vegetables and ornamentals worldwide. Fungicide treatments are effective for disease control, but bear the risk of resistance development. The major resistance mechanism in fungi is target protein modification resulting in reduced drug binding. Multiple drug resistance (MDR) caused by increased efflux activity is common in human pathogenic microbes, but rarely described for plant pathogens. Annual monitoring for fungicide resistance in field isolates from fungicide-treated vineyards in France and Germany revealed a rapidly increasing appearance of B. cinerea field populations with three distinct MDR phenotypes. All MDR strains showed increased fungicide efflux activity and overexpression of efflux transporter genes. Similar to clinical MDR isolates of Candida yeasts that are due to transcription factor mutations, all MDR1 strains were shown to harbor activating mutations in a transcription factor (Mrr1) that controls the gene encoding ABC transporter AtrB. MDR2 strains had undergone a unique rearrangement in the promoter region of the major facilitator superfamily transporter gene mfsM2, induced by insertion of a retrotransposon-derived sequence. MDR2 strains carrying the same rearranged mfsM2 allele have probably migrated from French to German wine-growing regions. The roles of atrB, mrr1 and mfsM2 were proven by the phenotypes of knock-out and overexpression mutants. As confirmed by sexual crosses, combinations of mrr1 and mfsM2 mutations lead to MDR3 strains with higher broad-spectrum resistance. An MDR3 strain was shown in field experiments to be selected against sensitive strains by fungicide treatments. Our data document for the first time the rising prevalence, spread and molecular basis of MDR populations in a major plant pathogen in agricultural environments. These populations will increase the risk of grey mould rot and hamper the effectiveness of current strategies for fungicide resistance management.

Exploring Mechanisms of Resistance to Respiratory Inhibitors in Field Strains of Botrytis cinerea, the Causal Agent of Gray Mold

ABSTRACT Respiratory inhibitors are among the fungicides most widely used for disease control on crops. Most are strobilurins and carboxamides, inhibiting the cytochrome b of mitochondrial complex III and the succinate dehydrogenase of mitochondrial complex II, respectively. A few years after the approval of these inhibitors for use on grapevines, field isolates of Botrytis cinerea, the causal agent of gray mold, resistant to one or both of these classes of fungicide were recovered in France and Germany. However, little was known about the mechanisms underlying this resistance in field populations of this fungus. Such knowledge could facilitate resistance risk assessment. The aim of this study was to investigate the mechanisms of resistance occurring in B. cinerea populations. Highly specific resistance to strobilurins was correlated with a single mutation of the cytb target gene. Changes in its intronic structure may also have occurred due to an evolutionary process controlling selection for resistance. Specific resistance to carboxamides was identified for six phenotypes, with various patterns of resistance levels and cross-resistance. Several mutations specific to B. cinerea were identified within the sdhB and sdhD genes encoding the iron-sulfur protein and an anchor protein of the succinate dehydrogenase complex. Another as-yet-uncharacterized mechanism of resistance was also recorded. In addition to target site resistance mechanisms, multidrug resistance, linked to the overexpression of membrane transporters, was identified in strains with low to moderate resistance to several respiratory inhibitors. This diversity of resistance mechanisms makes resistance management difficult and must be taken into account when developing strategies for Botrytis control.

Reduction in the sex ability of worldwide clonal populations of Puccinia striiformis f.sp. tritici.

Puccinia striiformis f.sp. tritici (PST), has so far been considered to reproduce asexually with until very recently no known alternate host, has a clonal population structure in the USA, Australia and Europe. However, recently, high genotypic diversity in Eastern Asia and recombinant populations in China has been reported. Variations in the ability for sexual reproduction could provide an explanation for such a geographical gradient in genotypic diversity. In order to address this hypothesis, we tested for the existence of a relationship between the ability to produce telia, sex-specific structures that are obligatory for sexual cycle, and the genetic diversity of populations measured using neutral markers, in a set of 56 isolates representative of six worldwide geographical origins. Clustering methods assigned these isolates to five genetic groups corresponding to their geographical origin, with eight inter-group hybrid individuals. Isolates representing China, Nepal and Pakistan displayed the highest telial production, while clonal populations from France and the Mediterranean region displayed very low telial production. The geographic cline in telial production corresponded to the gradient of genotypic diversity described during previous studies, showing a clear difference in telial production between clonal vs. diverse/recombinant populations. The higher mean Qst value (0.822) for telial production than the Fst value (0.317) suggested that telial production has more probably evolved through direct or indirect selection rather than genetic drift alone. The existence of high telial production in genetically diverse populations and its reduction in clonal populations is discussed with regard to evolution of sex, PST centre of origin and distribution of its alternative host.

Chemical Control and Resistance Management of Botrytis Diseases

Chemical control remains the easiest way to manage Botrytis epidemics on many crops. Nevertheless, actual concerns about the environment, human health and control sustainability invite to a smarter use of fungicides, aiming to delay resistance evolution in pathogen populations. This chapter deals with the mode of action of botryticides (including multi-site toxicants and molecules affecting specifically respiration, cytoskeleton, osmoregulation, sterol and amino-acid biosynthesis) and associated resistance cases, mostly due to target site modifications. We also present original resistance mechanisms for fungi such as detoxification and multidrug resistance. Finally, this chapter introduces strategies available to decrease selection pressure exerted by fungicides on Botrytis spp. populations with the long-term aim to improve resistance management in the field.

Diversity Within and Between Species of Botrytis

The genus Botrytis is highly diverse, with numerous species differing in terms of their biology, ecology, morphological features and host range. Progress in molecular genetics, and the development of relevant phylogenetic markers in particular, has resulted in the establishment of ≈ 30 species, a hybrid and a species complex. At least seven new species have been identified in the last decade, albeit with limited scientific support in some cases. B. cinerea has long been known to display broad population diversity, possibly due to intense recombination and large population sizes. The introduction of powerful markers, such as SSRs, has provided new insight into the respective contributions of the forces driving this diversity. It has recently been shown that populations may be structured, not only by the host plant as shown in preliminary studies, but also by other factors, such as cropping system, geography and fungicide applications. Evidence of recombination and gene flow, between and within compartments, has also been obtained. Finally, this chapter focuses on the biological and genetic characteristics of Botrytis spp. favouring their adaptation to their local environment and speciation. This information is particularly useful for improving the management of diseases on cultivated hosts.

Oilseed Rape Pathogens in France

In France, a dozen of diseases can affect rapeseed, but Sclerotinia stem rot is the major disease for which chemical control remains the main way to prevent severe attacks. Five fungicide modes of action are effective against Sclerotinia sclerotiorum and some solo or mixed products are registered in France. The history and current status of resistance to these different groups in French oilseed rape crops are presented. Strategies for preventing and managing fungicide resistance are mainly based on the alternation of products from different classes of biochemical modes of action.

Grapevine Gray Mold in France

Chemical control remains the main approach for decreasing the incidence of gray mold, a disease of many crops, including grapevine, caused principally by Botrytis cinerea. Fungicides with seven different modes of action are currently authorized in French vineyards, but specific resistance has developed against five of these modes of action. Multidrug resistance caused by fungicide efflux has been detected and affects all the classes of fungicide used. Here, we present the history and current status of resistance to the various botryticides in French vineyards. We also discuss resistance management options, based on decreases in the number of botryticide applications per season and the alternation of single products from different classes of molecules with different biochemical modes of action.